A membrane-regulated, sugar-phosphate: phosphohydrolase has been purified and characterized from Lactobacillus casei. Antibodies to the enzyme have been obtained for use in structure/function experiments. Lactose analogs containing non-metabolizable, (14C)-labeled 2-fluoro-D-glucose or 2-deoxy-D-glucose have been prepared enzymatically. Mutants of Streptococcus lactis have been isolated by positive resistance and UV mutagenesis procedures. These mutants lack ATP-dependent glucokinase and/or PEP-mannose: phosphotransferase activities and are incapable of growth on glucose. Through the use of the bacterial mutants, and transport studies with (14C)-2F-lactose and (14C) 2D-lactose, definitive evidence for intracellular phosphorylation of free sugars via the PTS has been obtained. Biochemical, and physiological studies with Peptostreptococcus anaerobius have revealed that proline is a requirement for the transport and metabolism of glucose by this anaerobic oral pathogen.